The field of the present invention relates to compositions and methods for treating or preventing disease in aquatic animals such as farmed fish (e.g. catfish or tilapia) and crustaceans (e.g., shrimp). In particular, the present invention relates to compositions and methods comprising or utilizing attenuated strains of Aeromonas hydrophila for treating or preventing diseases such as Motile Aeromonas Septicemia (MAS) in aquatic animals such as farmed fish.
Aquaculture is one of the fastest growing industries in the world. It is developing, expanding and intensifying in almost all regions of the world due to the vast global population demand of aquatic food products and the leveling-off of the capture fisheries. According to FAO, in 2010 alone, the World aquaculture production reached 60 million tons (excluding aquatic plants and non-food products), with an estimated, total value of US $119 billion. In 2010, 181 countries and territories had aquaculture production, with Asia accounting for 89 percent of world aquaculture production by volume, in which 61.4 percent was from China. During the last thirty years, the production of global food fish protein has expanded by almost 12 times, at a rate of 8.8 percent more per year (FAO 2012) According to FAO, global aquaculture production will need to achieve 80 million tons by 2050 just to maintain the current level of per capita consumption. It is not possible for the capture fisheries industry itself to meet this big global aquatic food need. Even with the fast growing rate and the significant contribution of the aquaculture industry, it is still a big challenge for the aquaculture to achieve this goal. (FAO 2005).
In United States, the aquaculture industry is dominated by finfish production (FAO 2012). Being the largest sector of the aquaculture industry, channel catfish farming produced more than 400 million dollars which accounts for approximately half of the total aquaculture production in U.S. in 2010, in the top 10 fish and seafood that consumed among Americans rank, Catfish raised by farm was sixth, about 0.8 pound per person per year. (Hanson & Sites, 2012). Most catfish are produced in the south of the United States and Mississippi, Alabama, Arkansas, and Texas are the top four States for catfish in the United States, which accounts for 94 percent of total sales (USDA, 2014).
Even though the U.S. catfish industry is the dominant aquaculture practiced in the United States, catfish production is vulnerable to adverse impacts of disease and environmental conditions. Before the 1990's, the strategy of management practices was ‘low-density’, which resulted in good pond water quality, lower overall stress on fish populations and less efficient pathogen transmission. However, due to the great competition from the Asian countries especially China, the producers applied much more intense production strategies, such as multiple batch cropping systems, higher stocking density, more feed put into the culture systems. All of these practices lead to the emergence of the infectious diseases which now becomes the primary limiting factor in catfish production. Disease outbreaks in recent years are very common even on efficient and well-built catfish farming facilities. According to MSU (Mississippi State University) reports, infectious diseases have caused approximately 45 percent of inventory losses on catfish fingerling farms, and about 60 percent of the overall catfish losses are attributed to single or mixed bacterial infections, 30 percent due to parasitic infection, 9 percent from fungal infection, and 1 percent result from viral etiology. Economic losses resulting from infectious diseases are believed to cost producers millions of dollars in direct fish losses each year. Furthermore, infectious diseases can also impact the profitability by increasing treatment costs, reducing food consumption by fish due to the flavor change and appearance, increasing feed conversion ratios, and causing harvesting delays.
The major bacterial diseases in catfish that affect the catfish industry are: Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri (Hawke, 1979); Motile Aeromonas septicemia (MAS), which is caused by Aeromonas species (Austin & Adams, 1996) and Columnaris (also referred to as cottonmouth) which is caused by Flavobacterium columnare (Wagner, 2002). The economic losses due to the ESC, according to USDA were about 30 to 50 million dollars each year (Shoemaker et al., 2007; USDA, 2010a, 2010b). The yearly losses caused by the Columnaris are estimated to be 30 million dollars (Declercq, 2013). MAS also causes huge amount of economic losses which are not limited to channel catfish but also including tilapia, catfish, goldfish, common carp, and eel (Pridgeon et al., 2011).
Prior to 2009, MAS in channel catfish caused by A. hydrophila was not a significant concern because the catfish aquaculture operations in the southeastern United States had not experienced a major outbreak (Hemstreet, 2010). However In 2009, catfish farmers in west Alabama reported severe disease outbreaks which were then proved to be caused by a highly virulent strain of A. hydrophila, ML09-119, to channel catfish (Ictalurus punctatus). From 2009-2011, Alabama catfish famers lost more than 7.5 million pounds of catfish that were market-size and estimated to be $3 million due to this epidemic strain of A. hydrophila (Pridgeon, 2011; Liles, 2011). It is reported that A. hydrophila epidemic strain, ML09-119, is highly virulent to channel catfish, causing severe mortality within 24 h post exposure with certain amount of dose (Pridgeon, 2011). The epidemic MAS outbreaks caused by Aeromonas hydrophila are so devastating that it is highly essential to investigate the virulence nature of this pathogen, identify the virulence related genes and create live avirulent bacterial mutants that are vaccine candidates for this bacterial disease. So far there are only a few factors identified for the A. hydrophila epidemic strain, and no commercial vaccine or treatment for the epidemic MAS are available right now. Three attenuated A. hydrophila vaccines were reported to offered 86-100% protection against their virulent parents at 14 days post vaccination (dpv), when the channel catfish were vaccinated with the mutants at dosage of 4×105 CFU/fish. These mutants were developed from the virulent 2009 West Alabama isolates through selection for resistance to both novobiocin and rifampicin (Julia and Klesius, 2011). But these antibiotic resistant mutants are spontaneous mutants that could more readily revert to a virulent strain compared to targeted, stable genetic deletions in gene(s) responsible for virulence.
Therefore, there is a need for a better understanding of the virulence of A. hydrophila in order for vaccine development to progress. Here, the inventors disclose methods for identifying virulence factors of A. hydrophila and producing attenuated strains of A. hydrophila that have been made deficient in one or more virulence factors.